Rubella was first described in Germany in the 18th century and is, therefore, often referred to as German measles. It is a highly contagious disease characterized by a general rash and a mild fever. Its clinical aspects were, for a long time, confused with other infections, including measles. The major risk associated with rubella infection occurs during pregnancy when severe damage to the fetus can result in deafness, cataracts, cardiac abnormalities and microencephaly.
The rubella virus, the etiologic agent of rubella, belongs to the Togaviridae family. It is a roughly spherical enveloped virus about 60 nm in diameter. Its genome consists of a single positive stranded RNA (10 Kb). The envelope contains lipoproteins derived from host infected cells, a non-glycosylated nucleocapsid protein--C(33K)--and two glycoproteins--E1(58K) and E2(42-47K). The latter, responsible for the hemagglutination activity of the rubella virus, are acylated and linked by disulfide bonds.
Three strains of rubella virus (Therien, Judith, M33) have been described and portions of their genomes have been sequenced (Frey et al., 1986, Virology 154, 228-232; Terry et al., 1988, Arch. Virol. 98, 189-197; Clarke et al., 1987, Nucl. Acids Res. 15, 3041-3057).
Although rubella can be diagnosed by inoculating infected materials (usually nasopharyngeal secretions) into susceptible cell cultures, the most widely used diagnostic tests are based on the hemagglutinating properties of its glycoproteins. In those assays ("HAI"), the presence in a serum sample of antibodies to a hemagglutinin prevents the virus from binding to red blood cells (usually from chicken blood) thus inhibiting hemagglutination (Peetermans and Huygelen, 1967, Presse Med. 75, 2177-2178 and Lennette and Schmidt, 1979, in "Diagnostic procedures for viral, rickettsial and chlamydial infections", 5th Ed., American Public Health Association Inc., Washington). In such assays an increase in HAI antibody titers reflects a recent infection.
Since the introduction of enzyme-labeled antibodies (Avrameas, 1969, Immunochemistry 6, 43-52), enzyme-linked-immunosorbent (ELISA) have been used for the diagnosis of a large variety of viral and bacterial infections, including rubella infections. Serodiagnosis of rubella infections using ELISA techniques, for example, was first described by Voller and Bidwell (1975, Br. J. Exp. Pathol. 56, 338-339).
In ELISA, viral extracts are typically coated onto the surface of plastic wells and antibodies (if any) in a serum sample or analyte are bound to the adsorbed proteins from the viral extracts. After appropriate washing, the presence of antibodies bound to the proteins in the well is detected using antibodies to human immunoglobulins conjugated to a signal, such as a horseradish peroxidase. After washing off unbound signal, the levels of enzymatic activity in each well are measured. Other forms and variations of ELISA are also well known and often used.
The introduction of ELISA for the specific determination of rubella IgM and IgG has been responsible for the rapid decline (i.e., from 45% in 1978-1980 to 19% in 1982) of HAI diagnostic assays for rubella viral infections (Steece et al., 1985, J. Clin. Microbiol. 21 (1), 140-142). Compared to HAI tests, ELISA requires no pretreatment of the sera and only one or two dilutions per serum sample. The amount of antigenic material used in ELISA is also smaller than what was required in the former HAI assays.
There are unfortunately several problems with presently used ELISA diagnostic tests for rubella infection. Specifically, internal variations between wells and between different batches of rubella antigens used to coat the wells are often observed. These variations are likely a consequence of various difficulties encountered in reproducibly isolating the rubella virus from host cell cultures.
The production of the rubella virus in tissue cultures is technically difficult. The virus grows to low titers, is difficult to separate from cellular membrane debris and is highly labile (Ho-Terry et al. 1986, Arch Virol. 87, 219-228; Chagnon and LaFlamme, 1964, Can. J. Microbiol. 10, 501-03). This makes it difficult to isolate the virus from cellular debris originating from host cells. In an attempt to overcome this problem, most ELISA techniques for detecting rubella infection employ a series of wells coated with extracts prepared with uninfected cells and another series of wells coated with extracts prepared from rubella-infected cells. Each serum sample is then tested on both series of wells and the net response is calculated by subtraction of the signal measured on the wells coated with extracts prepared with uninfected cells from the one measured on the wells coated with extracts prepared with rubella-infected cells.
Terry et al. (1988, Arch. Virol. 98, 189-197) and Ho-Terry et al. (1986, Arch. Virol. 90, 145-152 and European Patent Application No. 88306191.3) refer to the reactivity of three non-competing monoclonal antibodies directed against the rubella E1 glycoprotein. The epitopes bound by each of these monoclonal antibodies have been identified and are designated EP1, EP2 and EP3. Monoclonal antibodies directed to EP1 and EP2 exhibit both hemagglutination inhibition and neutralizing activity. Monoclonal antibodies to EP3 exhibit only neutralizing activity. The exact location of these three epitopes in the viral genome is shown in FIG. 1. None of these documents describes synthetic peptides corresponding to the EP1, EP2 or EP3 epitopes. It is thus not known how useful synthetic peptides corresponding to such epitopes might be in the development of a diagnostic test for rubella antibody detection.
The rubella pandemic of 1963-1965 prompted the development of a vaccine against rubella. (Parkman et al., 1966, N. Engl. J. Med. 275, 569-574). It was comprised of live attenuated viruses and is immunogenic in at least 95% of the recipients. While neutralizing antibodies appear later than following a natural infection and at levels as much as ten-fold lower, the vaccine-stimulated antibodies nonetheless effectively protect recipients from the disease. The present rubella vaccines, however, have some drawbacks. For example, a significant proportion of vaccinees suffer occasional arthritis (mainly seen in adult women), mild rash, fever and lymphadenopathy. Protection conferred by the vaccine also lasts for only 5-10 years, rather than the longer-lasting immunity that follows natural infection. Most importantly, small amounts of infectious virus typically appear in the nasopharynx 2-3 weeks after immunization, making vaccination very dangerous for pregnant women coming in close contact with a recently vaccinated person or even worse having herself been vaccinated while not knowing she was pregnant.
Vaccines based on synthetic or recombinant peptides would not present this hazard because the antigenic material would be totally innocuous. However, such vaccines are not now available and the immunogenicity and neutralizing properties of peptide-based vaccines are unknown. Furthermore, not all peptides are expected to be useful in vaccines. For example, high antibody titers in HAI tests do not correlate well with protection against rubella infection (Partridge et al., 1981, Br. Med. J. 282, 187-188). This may be due to the fact that epitopes involved in hemagglutination and neutralization are different (Trudel et al, 1982, J. Virol. Methods 5, 191-197). Diagnosis based on the detection of neutralizing antibodies, on the other hand, should have a high predictive value for immune status and prevention of rubella infection.
These differences are important, not only in evaluating peptide-based vaccines against rubella but in assaying the immune status of patients with respect to rubella infectivity. For example, the "purified" rubella antigens now available are potentially infective and carry both the hemagglutinating and neutralizing epitopes. Thus, specific tests for immune status using these antigens are questionable, and the antigens used in those vaccines may be infectious.
Considering these problems, we have selected certain peptide sequences on the E1 and C proteins of the rubella virus and prepared peptides defined by them. Our peptides selected for their ability to bind high levels of antibodies, as measured by an ELISA, are useful in diagnostic tests for rubella infection. Peptides of this invention recognized by neutralizing antibodies are also useful as the active ingredient of a totally innocuous rubella vaccine.